Polymorphonuclear neutrophils recognize conserved structures in microbial pathogens, referred to as pathogen-associated molecular patterns (PAMPs) by means of toll like receptors (TLRs), rendering them important players in the innate immune system1. Among the TLRs, TLR9 signaling has been implicated in the pathogenesis of lupus and other ‘autoimmune’ diseases2,3. TLR9 is exclusively expressed in intracellular vesicles and recognizes unmethylated Cytosine-phosphatidyl-Guanine (CpG) DNA motifs that are frequently present in bacteria and viruses, but are rare in mammalian cells1. TLR ligand binding activates the regulatory transcription factor nuclear factor-κB and induces the secretion of pro-inflammatory cytokines.
Trauma and other forms of cellular injury can release endogenous ‘damage’-associated molecular patterns (DAMPs) that, similar to microbial PAMPs, also activate innate immunity4. Upon such injury, cells can release mitochondrial DNA (mtDNA) which contains unmethylated CpG motifs and resembles bacterial DNA1. MtDNA in clinically relevant concentrations has been shown to activate human polymorphonuclear neutrophils through TLR9, a process which promotes their degranulation, and elicits systemic inflammation and organ injury including inflammatory lung injury4.
A recently discovered aspect of the antimicrobial defense of polymorphonuclear leukocytes is their ability to release chromatin in the form of extracellular fibers called neutrophil extracellular traps (NETs)5-7. It has been demonstrated in eosinophils that the process of NET formation is an early event of cell death called ‘NETosis’. Polymorphonuclear neutrophils and eosinophils play an important role in the pathogenesis of ANCA-associated vasculitis (AAV), such as Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CSS). These forms of vasculitis are associated with the presence of antineutrophil cytoplasmatic antibodies (ANCA) in the serum of affected patients8. It has been demonstrated that in WG, ANCA are mostly directed against proteinase 3 (PR3), whereas in MPA ANCA tend to be directed against myeloperoxidase. PR3-ANCA may have a direct pathogenic role in the pathogenesis of WG by activating primed neutrophils and also by inducing endothelial cell damage8.
It has been recently demonstrated that endothelial cell activation elicits NET formation and that conversely NETs can induce endothelial cell injury9. Thus NETs may also play an important role in the pathogenesis of AAV. In this context it is also interesting to note that the propensity for disease relapses in WG may be enhanced by bacterial infections with Staphylococcus aureus10-12, which are also known to trigger NET formation13. Finally, it has been shown that not only bacterial stimuli such as S. aureus, but also ANCA are able to induce NETosis14. Indeed PR3 and myeloperoxidase have been found in physical association with NET components in the glomeruli of individuals with ANCA-associated glomerulonephritis14.
A problem of the present invention is to provide a suitable marker useful in supporting or decreasing the likelihood of the diagnosis of AID in clinically unclear situations. Another problem of the present invention is to provide a marker of disease activity that could be useful in determining the intensity of the therapy of said AID.
The inventors surprisingly found that patients suffering from AID show elevated levels of mitochondrial DNA circulating in plasma. Furthermore, the ratio of mitochondrial DNA to nuclear DNA was elevated. The invention therefore pertains to the diagnosis of AID by determining the amount of mitochondrial nucleic acids and/or the ratio of mitochondrial to nuclear nucleic acids, and to other aspects associated therewith.
Zhang et al. (2010) Nature 464, 104-108 describes experiments suggesting that circulating mitochondrial ‘damage’-associated molecular patterns (DAMPs) cause inflammatory responses to injury. Zhang et al. does not disclose the diagnosis of autoimmune and autoinflammatory diseases (AID).
Khurana et al. (2008) Invest. Ophthalmol. Vis. Sci. 49, 3299-3304 investigates whether oxidative mitochondrial DNA damage occurs early in experimental autoimmune uveitis (EAU), before leukocyte infiltration. Damage to mitochondrial DNA and nuclear DNA was assessed using a long quantitative polymerase chain reaction technique. Khurana et al. does not disclose the diagnosis of autoimmune and autoinflammatory diseases (AID) nor does it suggest that elevated levels of mitochondrial DNA in plasma could be indicative of AID.
Yu et al. (2009) Genes and Immunity 10, 601-605 reports an association of a certain mitochondrial gene polymorphism (UCP2-866 G/A) with chronic inflammatory diseases. Yu et al. does not determine the amount or concentration of circulating mitochondrial DNA in samples of patients. Rather, the disclosure of Yu et al. is limited to genotyping of the polymorphism.
Feng et al. (2000) Chinese Medical Journal 113(8), 743-746 investigates the primary mutations of mitochondrial DNA associated with Leber's hereditary optic neuropathy (LHON) in patients with optic neuropathy. Feng et al. does not determine the amount or concentration of circulating mitochondrial DNA in patient samples. Rather, Feng et al. detects certain mutations in mitochondrial genes.
WO 2007/106790 A2 describes methods, compositions and uses thereof, relating to vitiligo or vitiligo-associated autoimmune/autoinflammatory disease (VAAAD). In particular embodiments, genetic variations in the NALP1 gene are of use to detect, diagnose, predict the risk of or treat at least one of vitiligo or VAAAD. In more particular embodiments, the presence of genetic variations such as single-nucleotide polymorphisms (SNPs) in NALP1 genetic region are of use to detect, diagnose or predict the risk of VAAAD. In other embodiments, inhibitors targeted to NALP1, caspase-1 or caspase-5, ASC (PYCARD), interleukin-1ss, interleukin-1ss receptor, or interleukin 18 may be administered to a subject to treat VAAAD. The gene NALP1 is a chromosomal gene, and, therefore, WO 2007/106790 A2 does not aim at determining the amount of mitochondrial DNA.
EP 812922 A2 describes novel human mitochondrial polymorphisms, and probes and primers for detecting the same. Detection of such polymorphisms is said to be useful in a variety of fields such as forensic analysis, epidemiology and preventive medicine. This reference does not determine the amount or concentration of circulating mitochondrial DNA in patient samples. Rather, the disclosure of EP 812922 A2 et al. is limited to genotyping of the polymorphisms.
EP 1538218 A1 describes methods to diagnose or screen for inflammatory condition or disease, including auto-inflammatory disease and affective disorder, in a subject, preferably a human subject, by assaying for a marker for an inflammatory disease. Described is a method to diagnose or screen for an inflammatory disease in a subject, said method comprising determining the level of various inflammatory-specific gene product(s) in a biological sample isolated from said subject, preferably peripheral blood monocytes, wherein said inflammatory-specific gene is selected from the group comprising HSPC228, 34703_f_at, MCP-3, CCL2, EMP1, CDC42, TLE3, SPRY2, p40BBP, HSPC060, NAB2, HSPA1A, HSPA1B, MAPRE2, OAS1, CCR2, CX3CR1, DOK1, HBB, G-gamma globin, THBD, PHLDA1, DTR and GNLY. EP 1538218 A1 neither teaches nor suggests determining the amount of mitochondrial DNA.